1. Field of the Invention
The invention relates to a method for detecting genetic variations in bovine which influence their susceptibility to bovine spongiform encephalopathy.
Transmissible spongiform encephalopathy (TSE) agents or prions induce fatal neurodegenerative diseases in humans and in other mammalian species. They are transmissible among their species of origin, but they can also cross the species barrier and induce infection and/or disease in other species. Human TSEs include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, Kuru and fatal familial insomnia (36). In animals, 4 distinct TSE diseases are recognized: scrapie in sheep and goats, transmissible mink encephalopathy (TME) in mink, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE) in cattle. BSE was transmitted via BSE-contaminated feed to cats (feline spongiform encephalopathy, FSE) and exotic ungulates (exotic ungulate encephalopathy, EUE) and via contaminated food to humans (48, 49, Smith et al., 2004. CTMI 284: 161). BSE first emerged in the United Kingdom in 1986, and has subsequently spread to many countries, predominantly in Western Europe. These outbreaks, caused by the consumption of infected meat and bone meal containing a malformed protein, have resulted in the destruction of thousands of cattle and have caused significant economic losses.
2. Description of the Prior Art
Prions are proteinaceous infectious particles and are the causative agents of TSEs. They are host coded proteins that have undergone conformational changes and have biological and physicochemical characteristics that differ significantly from those of other infectious agents. For example, they are resistant to inactivation processes that are effective against conventional viruses including those that alter nucleic acid structure or function. These include ionizing and UV radiation (1) or inactivation by formalin (20). In addition, infectivity is highly susceptible to procedures that modify protein conformation. Protein denaturants are effective at reducing infectivity titers but complete inactivation requires extremely harsh conditions, such as up to 4 hours of autoclaving at 134° C. or treatment with 2 N NaOH (39). In TSE disease, the normal cellular protein, PrPC, is converted to abnormal prion protein, PrPSc. PrPSc exhibits increased beta sheet content, a change that may drive the additional changes in solubility and protease resistance (38). Unlike normal cellular protein, PrPSc is relatively insoluble in detergents, is relatively resistant to proteases (37) and is capable of causing a conformational change in additional molecules of PrPc. The precise function of the normal PrPc in healthy animals remains unknown. PrPc might play a role in sleep physiology, in resistance to oxidative stress, in signal transduction and in self-renewal of hematopoietic stem cells (16, 29, 31, 53).
TSE disease involves the accumulation of PrPSc in the central nervous system (CNS) of the host, eventually leading to neurodegeneration and disease. In TSE-affected animals, PrPc has a determinant role in the incubation time and species barrier (8). Transgenic mice lacking prion protein gene (Prnp) expression are not susceptible to TSE agents or prion infection, demonstrating the key role of PrP in TSEs (8). Susceptibility to prions thus depends upon the presence of PrPc on the cell membrane of the host; prions do not propagate in brains that lack PrPc (6).
Widely referred to as “mad cow disease”, BSE was first identified as a TSE of cattle in the mid 1980s in the U.K. and more than 180,000 positive cases have been diagnosed in the U.K. to date. BSE is a chronic degenerative disease affecting the central nervous system of cattle. Affected animals display changes in temperament, abnormal posture, incoordination and difficulty in rising, decreased milk production, and/or loss of body weight despite continued appetite (40). The average incubation period is about 4-6 years and all affected animals succumb to the disease (28). Following the onset of clinical signs, the animal's condition deteriorates until it either dies or is destroyed. This process usually takes from 2 weeks to 6 months. Most cases in Great Britain occurred in dairy cows between 3 and 6 years of age with the highest susceptibility to infection being in the first 6 months of life; adult cattle are at relatively low risk of infection (3).
Using epidemiological surveillance programs, many European and non-European countries have discovered BSE-positive animals within the last decade (17, 34). Validated diagnostic tests for BSE require brain tissue (33, 47). There are no validated ante mortem tests for BSE available at present. The original diagnostic test method was histopathology in which sections of brain were examined under a microscope, and the classical vacuoles and spongiform changes in specific areas of the brain would allow a diagnosis (33). In the mid-1990s, immunohistochemistry (IHC) and Western blotting were developed which allowed the detection of PrPSc in tissues (33). Both IHC and Western blot are considered confirmatory tests for BSE by the World Organization for Animal Health-OIE (33). In the past decade, so-called “rapid tests” have been introduced commercially for BSE surveillance (33).
However despite these and other advances, the need remains for improved methods for diagnosing BSE infected animals, and particularly for detecting bovine animals having increased susceptibility to BSE.